The identical gain-of-function JAK2V617F allele is present in the majority of patients with the myeloproliferative disorders (MPD) polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), and in vitro and in vivo data demonstrate the central role of this activating mutation in the pathogenesis of these disorders. Although small molecule inhibitors of JAK2 kinase activity are being developed, these compounds inhibit both wild-type and mutant JAK2 kinase activity. The central role of JAK2 signaling in a spectrum of cellular processes suggests these compounds may have hematopoietic and non-hematopoietic toxicities. The aims in this proposal are designed to implement a high throughput screen for mutant selective inhibitors of the JAK2V617F kinase, and to optimize the probes from this screen using secondary assays. The aims of this proposal are: A. Develop, transfer, and screen a HTS assay designed to identify inhibitors of the JAK2V617F mutant kinase. B. Perform a counter screen using Ba/F3-EPOR-JAK2 cells grown in the presence of EPO in order to identify compounds that are selective for the JAk2V617F mutant kinase. Test the compounds in secondary assays and optimize if necessary to generate a chemical probe suitable for Aim C. C. Characterization of JAK2V617F mutant select inhibitors using in vitro and in vivo assays, in order to demonstrate proof of concept for efficacy for mutant selective kinase inhibition, followed by optimization and preclinical development of JAK2V617F inhibitors that demonstrate activity in cell culture and animal model systems. The identification of mutant selective inhibitors is of biologic and clinical importance, as this would provide a novel approach to the design of molecularly targeted therapies and would offer a way to develop candidate compounds with an improved toxicity profile for MPDs and for human malignancies in general. [unreadable] [unreadable] [unreadable]